Chemosphere 177 (2017) 77e83

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Chemosphere
journal homepage: www.elsevier.com/locate/chemosphere

Agricultural pesticides and veterinary substances in Uruguayan

beeswax

a, Mauricio Grajales b, Christophe Lhe
Jorge Harriet a, Juan Pablo Campa
ritier c,
Antonio Go
mez Pajuelo d, Yamandú Mendoza-Spina e, Leonidas Carrasco-Letelier f, *
a
n Apicultura, Direccio
Seccio
n de Laboratorios Veterinarios, Ministerio de Ganadería, Agricultura y Pesca (MGAP), Ruta 8 Brig. J. A. Lavalleja Km 17500,
Montevideo, Uruguay
b
Apícola Integral Las Piedras, Canelones, Uruguay
c
n de Exportadores de Miel, Uruguay
Asociacio
d
Pajuelo Consultores Apícolas, Sant Miquel 14, 12004 Castello
n, Spain
e
Beekeeping Unit, National Agricultural Research Institute (INIA), Uruguay
f
Production and Environmental Sustainability, National Agricultural Research Institute (INIA), Uruguay

h i g h l i g h t s g r a p h i c a l a b s t r a c t

Lipophilic xenobiotics was accumu-

lated in recycled beeswax (RB).

Xenobiotic pollution presented a
higher frequency and concentration
in RB.

Some fungicides and neonicotinoids
could have synergistic effects.

The use of honey cappings to make
beeswax foundation will reduce the
hive health risk.

a r t i c l e i n f o a b s t r a c t

Article history: Over the last decade, Uruguay has expanded and intensified its rainfed crop production. This process has
Received 4 November 2016 affected beekeeping in several ways: for example, by reducing the space available. This has increased the
Received in revised form density of apiaries, the risk of varroosis and acaricide use. Additionally, the dominance of no-tillage crops
10 February 2017
has increased the frequencies of application and of loads of pesticides in regions where such crops share
Accepted 25 February 2017
the land with beekeeping and honey production. Therefore, the exposure of bees to xenobiotics (agri-
Available online 28 February 2017
cultural pesticides and veterinary products) has increased in line with pollution of hives and their
Handling Editor: Caroline Gaus products. To document pollution from hive exposure to pesticides, we surveyed the presence of 30
xenobiotics normally used in Uruguay, in recycled beeswax (RB) and in honey cappings (HC) from the
2010 MSC: main Uruguayan beekeeping regions. There was contamination of all the analyzed samples (RB and HC)
00-01 with the herbicide atrazine at a range of 1e2 ng g1. At least three or four additional xenobiotics were
99-00 detected: insecticides (chlorpyrifos-ethyl and thiacloprid); fungicides (azoxystrobin and tebuconazole);
and veterinary products (coumaphos, ethion, and tau-fluvalinate). The frequency of detection of
Keywords:
Honey bee chlorpyrifos-ethyl and coumaphos in RB samples was higher than in those of HC. Moreover, the con-
Pesticides centrations of azoxystrobin, coumaphos, and tebuconazole in RB samples were higher than in HC
Acaricides
Beeswax
Hive health

* Corresponding author.
E-mail addresses: jharriet@mgap.gub.uy (J. Harriet), lcarrasco@inia.org.uy
(L. Carrasco-Letelier).

http://dx.doi.org/10.1016/j.chemosphere.2017.02.131
0045-6535/© 2017 Elsevier Ltd. All rights reserved.
78 J. Harriet et al. / Chemosphere 177 (2017) 77e83

samples. Therefore, we suggest the use of HC to produce recycled printed beeswax films for use in hives
to minimize pollution transfer.
© 2017 Elsevier Ltd. All rights reserved.

1. Introduction 2. Materials and methods

Agriculture on the Atlantic side of South America is tied to global 2.1. Study zone
commodity prices (Graesser et al., 2015). This form of agriculture is
based on achieving maximal biomass production through reducing The expansion and intensification of Uruguayan crop cultivation
or eliminating natural constraints, with the use of pesticides as one has mainly been in the Western Departments (a Department is an
of the main tools. In this regional framework, Uruguay has under- administrative division of our national territory). Historically, the
gone recent intensification and expansion of its rainfed areas, with same regions have been used for beekeeping and honey produc-
a land-use change by the introduction of glyphosate-resistant tion; thus, in some areas, there are more than five hives per km2
soybeans as the foremost crop grown in rotation systems (Harriet and Campa
, 2014). For these reasons, the beeswax used in
(Ce
spedes-Payret et al., 2009). Thus, the Uruguayan rainfed crops this study was collected from Departments with prominent agri-
increased from 278,000 ha (ha) in 2004 to 1.334 million ha in 2015 cultural and honey production (Fig. 1).
(DIEA, 2016). This land-use change led to the growth of annual The beeswax samples were obtained in 2014 from eight bee-
imports of formulated pesticides from 14,326 Mg in 2004 to keepers and two Uruguayan beeswax companies (Apícola Integral
24,160 Mg in 2015 (Servicios agrícolas, 2016) and increased the load Las Piedras and TELGAR). Each beekeeper supplied one sample of
and frequency of pesticide use. This has increased pesticide expo- HC and another of RB. The beeswax recycling companies provided
sure and the risk of pollution and toxic effects on pollinators and four samples of HC and eight samples of RB. In this last case, the RB
other nontargeted organisms (Ca
rcamo, 2010; Carrasco-Letelier was made with a mixture of beeswax from different beekeepers.
et al., 2006; Pareja et al., 2011). Additionally, zones free of pesti-
cides were reduced in agricultural regions and thus restricted the
2.2. Determination of pesticides and veterinary products
pollen diversity available for beekeeping. These factors promoted
increases in apiary density (Harriet and Campa
, 2014), in the hive
The xenobiotics (pesticides and veterinary products) were
health risk of varroosis contagion (Anido et al., 2015) and in the
extracted using published protocols (Niell et al., 2014). Briefly, this
frequency of acaricide application for controlling varroosis. More-
procedure consists of liquidliquid partitioning (acetonitrile:
over, such xenobiotic exposure can be propagated and increased
melted wax) followed by freeze-out and primarysecondary amine
through the use of beeswax foundations. Xenobiotics can be
with C18 dispersive solid phase extraction cleanup. The extracted
transferred directly via beeswax by its application to new combs
xenobiotics were determined using liquid chromatogra-
(e.g., coumaphos) (Van Buren et al., 1992) and residues can be
phytandem mass spectrometry (LCMS/MS) and gas chroma-
retained by beeswax (Tremolada et al., 2004). Van Buren et al.
tographymass spectrometry (GCMS).
(1992) described this pollution in beeswax obtained commercially
LCMS/MS was performed with an Agilent 1200 LC system
in Europe. Since that report, such pollution has continued in
(Agilent, Quantum Analytics Inc., Foster City, CA, USA) and insert
different European countries (Bogdanov et al., 2003; Persano Oddo
coupled to a 4000 105 QTRAP® LC/MS/MS System from AB SCIEX
et al., 2003; Ravoet et al., 2015), USA (van Engelsdorp et al., 2009)
(Foster City, CA, USA) run in the scheduled MS/MS-mode. LC sep-
and, recently, in Chile (Neira et al., 2011). Such beeswax pollution
aration was performed as described by Niell et al. (2015) on a
could cause problems for the immune system of bees (Prisco et al.,
ZORBAX Eclipse XDB-C18 (150 mm  4.6 mm, 5 m) column (Agilent
2013), reduce the life expectancy of newborn honey bees (Orantes-
Technologies). The operation of the LC gradient involved the
Bermejo et al., 2010), affect honey bee product quality (Bogdanov,
following elution program: A, water/HCOOH 0.1% (v/v); and B,
2006) and, perhaps, lead to colony collapse disorder (CCD) (van
MeCN. This was run at 600 mL min1 starting with 10% component B
Engelsdorp et al., 2009).
at injection time for 1 min and gradually changing to 100% B over
In this way, both the increased exposure to xenobiotics in
15 min. This mobile phase was held for 10 min and then shifted
Uruguay, and beeswax pollution in similar agricultural conditions
back to the starting conditions (10% component B) and kept there
allow us to propose as a first hypothesis that Uruguayan hives
until 35 min after the initial injection with a volume of 5 mL. MS/MS
might be contaminated along with their beeswax. As a second
detection was performed in the multiple reaction monitoring
hypothesis, it could be argued that pollution in beeswax will
(MRM) mode using an electrospray ionization interface in the
increase in frequency and magnitude by the production of
positive ion mode. The ionization voltage was 4500 V, the nebulizer
beeswax foundation. This could lead to higher levels of xenobi-
gas was synthetic air at 70 psi, and the curtain gas was nitrogen at
otics in recycled beeswax (RB) than in the non-recycled waxes
30 psi. The solvent evaporation in the source was assisted by a
such as honey cappings (HC). To test these hypotheses, we
drying gas (heated synthetic air at 425  C and 50 psi). The optimal
assessed the presence of 30 different xenobiotics (pesticides and
MRM transitions, collision energies, and declustering potentials for
veterinary products) in HC and RB samples from the main Uru-
each investigated compound were determined by infusing stan-
guayan regions of honey bee production. Based on these results,
dard solutions with a syringe directly the to the instrument at a
we compared the frequencies and levels of pollutants in each
constant flow rate.
kind of wax to assess the consequences of each hypothesis. We
GCMS analyses were performed using an HP 6890 GC system
also estimated whether the concentrations might become a risk
coupled with a HP 5973 MS supported by reference libraries,
for bees.
equipped with an HP-5 (5% diphenyl 95% dimethylsiloxane) bonded
fused-silica capillary column (30  0.25 mm i.d.  0.25 m film
thickness; Hewlett Packard, Wilmington, DE, USA). Electron impact
 J. Harriet et al. / Chemosphere 177 (2017) 77e83
Fig. 1. (A) Uruguay in South America. (B) Rainfed crops (gray shading) located in the various Departments of Uruguay (outlined) and (C) Departments providing samples (gray
polygons).
mass spectra were obtained at 70 eV and monitored from 50 to
550 m/z in full scan mode for the selection of ions for analysis. The
MS system was programmed in a selected ion monitoring (SIM)
mode for analyses. The working parameters were: injector tem-
perature 280  C; interface temperature 280  C; carrier gas He,
constant flow at 1 mL min1; oven conditions from 120  C initial
(5 min hold), increased to 190  C at a rate of 10  C min1 (1 min
hold), then to 250  C at 5  C min1 (5 min hold), then to 300  C at
5  C min1 (5 min hold). The injection volume was 1.0 mL. The
identification of the compounds was confirmed by the injection of
matrix-matched standards and comparison of their retention times
and relevant MS ratios. The methods of validation and analytical
€m et al.
quality control requirements followed the criteria of Pihlstro
(2016).
The procedures and analysis were conducted by the analytical
services of the Department of Chemistry from CENUR-Noroeste
(Universidad de la República, Paysandú, Uruguay). Thirty different
xenobiotics were assayed, with the limit of quantification (LOQ)
reported for each (Table 1).
2.3. Data analysis
The frequency of positive detections of pesticides was analyzed
by Fisher's test; the comparison of the rates for independent
samples with n < 30. Comparisons of the median xenobiotic con-
centrations between HC and RB samples were done using
nonparametric Mann-Whitney tests. All tests were performed us-
ing R software (R Core Team, 2016) on a GNU/Linux 64-bit oper-
ating system (Ubuntu, 2016).
2.4. Standard risk approach
Risk assessment was done following the proposal of Sanchez-
Bayo and Goka (2014) (Eq. 1). To estimate the risk of bees affected
by contact with contaminated RB, we considered the worst sce-
nario: the first 15 days of bee life in which the bees are in direct
contact with wax. The residue dose was the product of the xeno-
biotic concentration in RB multiplied by 250 mg of wax (estimated
mass for a bee cell of 4.9 mm i.d.; 12 mm long; 1 mm wall thickness;
1.5 mm base thickness, and 0.961 g cm3 in density).
79
 
frequency ½%  residue ng g 1  0:25 ½g  15 ½days
Risk ð%Þ ¼  
LC50 ng bee1
(1)
3. Results
From the 30 xenobiotics surveyed, eight (atrazine, azoxystrobin,
chlorpyrifos-ethyl, coumaphos, ethion, tau-fluvalinate, tebucona-
zole, and thiacloprid) were detected in both RB and HC samples
(Table 2). Only pollution with chlorpyrifos-ethyl and tebuconazole
had significantly higher detection rates in the RB than in the HC
samples (Table 2).
Xenobiotic concentrations were compared using Mann-
Whitney nonparametric tests because the data were not normally
distributed or lacked homogeneity of variance. Moreover, for this
statistical analysis, all the values tagged as <LOQ were assumed to
have a value of LOQ/10. Analysis showed that the median concen-
trations of azoxystrobin, coumaphos and tebuconazole were higher
in RB than in HC samples. For ethion, tau-fluvalinate, and thiaclo-
prid, differences were not tested statistically because fewer than
three samples were available. Notably, atrazine pollution of
beeswax did not differ between RB and HC samples (Table 3).
The estimation of damage probability or risk for the honey bees
exposed to contaminated RB during their first 15 days of life
showed that the highest risk was posed by chlorpyriphos-ethyl and
coumaphos. The rest of the pollutants in beeswax seem not to pose
important risks for bees (Table 4).
4. Discussion
The results confirmed our first hypothesis, because all the
beeswax samples studied showed pollution with several agricul-
tural pesticides as well as veterinary substances (Table 2). Most of
this pollution was within ranges reported by other studies (Table 5)
and mainly with the pesticide profile reported by Mullin et al.
(2010) in terms of the pesticides atrazine, azoxystrobin, ethion,
and thiacloprid. Some of them were not reported as pollutants of
beeswax until the papers by Mullin et al. (2010) and Johnson et al.
(2010).
80 J. Harriet et al. / Chemosphere 177 (2017) 77e83

Table 1 respectively (Table 5).

Pesticides and veterinary products surveyed in this study. The limits of quantifica- Our risk analysis (Table 4) showed that the most important xe-
tion (LOQ) values are expressed in ng g1 of beeswax and analytical methods were
utilized for determinations.
nobiotics needing management are chlorpyrifos-ethyl and couma-
phos. These risk values became more relevant when we calculated
Chemical compound CAS number Purpose LOQ Analytical method the time needed to reach the LD50: called T50 by Sanchez-Bayo and
Acetamiprid 135410207 I 1 LCeMS/MS Goka (2014). The T50 value is the ratio between the LD50 and the
Atrazine 1912249 H 1 LCeMS/MS daily dose. This estimation showed that it only needed 6 days to
Azoxystrobin 131860338 F 0.1 LCeMS/MS
reach the LD50 for chlorpyrifos-ethyl. This result highlights the need
Betacyfluthrin 68359375 I 100 GCeMS
Boscalid 188425856 F 100 LCeMS/MS for environmental management to control the use of chlorpyrifos-
Bromopropylate 18181801 A 100 GCeMS ethyl and reduce its exposure to hives, because 6 days is easily
Carbaryl 63252 I 100 LCeMS/MS reached by honeybee larvae. The current pollution pattern would
Carbendazim 10605217 F 10 LCeMS/MS
probably damage hive conditions, because thiacloprid (a neon-
Chlorfenvinphos 470906 I 100 GCeMS
Chlorpyrifosethyl 2921882 I 100 GCeMS
icotinoid) and tebuconazole (a fungicide) have potential synergistic
Clothianidin 210880925 I 1 LCeMS/MS effects (Iwasa et al., 2004) in that the toxicity of such insecticides to
Coumaphos 56724 A 100 GCeMS bees is increased by the action of the fungicide. Furthermore, there
Cypermethrin 52315078 I 100 GCeMS are potential sublethal effects in terms of immune suppression, and
Deltamethrin 52918635 I 100 GCeMS
susceptibility to diseases (Prisco et al., 2013), such as reductions in
Diazinon 333415 I 100 GCeMS
Dimethoate 60515 I 10 LCeMS/MS queen weights, abnormalities and atypical behavior from exposure
Ethion 5631202 I 100 GCeMS to coumaphos and tau-fluvalinate (Haarmann et al., 2002). In this
Fipronil 120068373 I 100 GCeMS scenario, perhaps the worst outcome is the presence of thiacloprid,
Haloxyfoppmethyl 69806402 H 1 LCeMS/MS because such neonicotinoids have been reported as a source of many
Hexythiazox 78587050 A 100 LCeMS/MS
Imidacloprid 13826413 I 10 LCeMS/MS
problems for beekeeping and are potentially linked to CCD
Iprodione 36734197 F 200 LCeMS/MS (Farooqui, 2013; Krupke et al., 2012).
Methidathion 950378 I 100 GCeMS These results show five situations that must be checked at
Methomyl 16752775 I 10 LCeMS/MS future: first, warn about the risk for beekeepers that work to crops
Parathionmethyl 298000 I 100 LCeMS/MS
that use chlorpyriphos (e.g. soybean, sorghum, corn, apple, peach,
Pyraclostrobin 175013180 F 10 LCeMS/MS
Taufluvalinate 102851069 A 100 GCeMS tomato); second, the pollution of wax with coumaphos confirmed
Tebuconazole 107534963 F 0.1 LCeMS/MS the persistence, that agree with Tremolada et al. (2004) and Van
Thiacloprid 111988499 I 10 LCeMS/MS Buren et al. (1992), and show the need to reduce this accumula-
Thiamethoxam 153719234 I 10 LCeMS/MS tion by the use of RB; third, become need to review the current
Key: A, acaricide; F, fungicide; H, herbicide; I, insecticide. national systems for controlling agricultural pesticides and veteri-
nary products, because any difference in the LD50 of bees, like
Carrasco-Letelier et al. (2012) showed for this region, could increase
Differences in detection frequencies between HC and RB sam- the R(%) or T50 values. A risk condition, at least share with the
ples were only found for chlorpyrifos-ethyl and tebuconazole. The Rolling Pampas region, that includes Uruguay, Argentina, and the
detection frequencies did not show significant differences when south of Brazil (Carriquiriborde et al., 2014). Fourth, the current
the xenobiotics were ubiquitous (e.g., atrazine and coumaphos) or toxicological value do not offer any protection to native pollinators,
scarce (e.g., ethion, tau-fluvalinate, and thiacloprid) in the studied which imply bigger toxic effects; either by increased consumption
samples. Moreover, comparisons of absolute xenobiotic concen- of contaminated sources, as in the case of bumble bees showed by
trations in HC and RB samples were more sensitive than for fre- Sanchez-Bayo and Goka (2014) or because colonies of pollinators
quencies, because the RB samples had significantly higher have fewer resources to resist poisoning (Alix et al., 2014).
concentrations of azoxystrobin, coumaphos, and tebuconazole Finally, although the Pareja et al. (2011) and our work reports
(Table 3). In these examinations, although chlorpyriphos-ethyl and pesticide pollution patterns similar to those studied in regions with
thiacloprid showed no differences in concentrations between HC colony collapse disorder (CCD) (van Engelsdorp et al., 2009; Mullin
and RB samples, the levels were higher than those reported pre- et al., 2010), we still do not have any report that the CCD is being
viously by Chauzat and Faucon (2007) and Mullin et al. (2010), developed in Rolling Pampas Region.

Table 2
Number of positive samples of xenobiotics found in recycled beeswax (RB) and honey cappings (HC), as well as the results of Fisher's tests.

Xenobiotic Beeswax Number of positive samples Specific frequency (%) P by Fisher's test

Atrazine RB 16 100 1.000

HC 12 100
Azoxystrobin RB 10 63 0.169
HC 5 42
Chlorpyrifos-ethyl RB 12 75 0.011
HC 3 25
Coumaphos RB 16 100 0.429
HC 11 92
Ethion RB 2 13 0.158
HC 3 25
Tau-fluvalinate RB 1 6 0.508
HC 1 8
Tebuconazole RB 16 100 0.024
HC 8 67
Thiacloprid RB 2 13 0.439
HC 1 8
 J. Harriet et al. / Chemosphere 177 (2017) 77e83 81

Table 3
Median, number of samples and range of xenobiotics in beeswax samples: recycled beeswax (RB) and honey cappings (HC). All concentrations are expressed in ng g1.

Xenobiotic HC Median [min.-max.] RB Median [min.-max.] Mann-Whitney U test P

Atrazine 1 1 0.75 0.3865

[1ae2] [1e2]
Azoxystrobin 0.7 1 4.04 0.0443
[0.4e1] [0.4e7]
Chlorpyrifos-ethyl 100 10 1.69 0.4631
[10ae100] [10ae200]
Coumaphos 200 800 11.50 0.0007
[100e800] [500e2900]
Ethion 100 100
[10ae10a] [100100]
Taufluvalinate 100 100
Tebuconazole 2 3 9.13 0.0025
[1e3] [1e30]
Thiacloprid 12 10
[10e10]
a
Equivalent value to the LOQ, divided by 10 to obtain the estimated limit of detection.

Table 4
Risk (% probability) of contact exposure with 250 mg of polluted RB for 15 days and contact 48 h-LD50 values (ng per bee).

Xenobiotic Mean concentration in RB (ng g1) Acute contact 48 h LD50 (ng per bee) Risk (%)
a
Chlorpyrifos-ethyl 41.7 59 198.8
b
Coumaphos 1120.0 20,000 21.0
b
Tau-fluvalinate 100.0 8700 0.3
a,c
Ethion 100.0 20,600 0.2
a
Thiacloprid 10.0 38,820 1:2  102
a
Tebuconazole 4.8 2105 9  103
d
Atrazine 1.1 1105 4:1  103
a
Azoxystrobin 1.8 2105 2:2  103

References.
a
Acute contact toxicity (PPDB, 2016).
b
Sanchez-Bayo and Goka (2014).
c
Unknown mode of acute toxicity (PPDB, 2016).
d
BCPC (2016).

Table 5
Xenobiotic residues in beeswax of this study and reported in the literature. All values are expressed in ng g1.

Xenobiotic Class Determined Reported Reference

Means: HC; RB LOD min.-max. LOD

Atrazine H 0.9; 1.1 0.1 1e31 1 f

Azoxystrobin F 0.7; 1.8 0.01 1e278 1 f
Chlorpyrifos-ethyl O 70; 41.7 10 7.1e19 5 b
172 6 g
1e890 0.1 f
3e751 a
Coumaphos O 336; 1120 10 >LOD-4113 5 b
46e380 NR d
1e91,900 1 f
826e6312 NR e
7e150 a
Ethion O 10; 100 10 84e131 2 f
Tau-fluvalinate P 100; 100 10 15e422 5 b
155e965 NR d
2e204,000 1 f
27e88,659 19e21 g
104e204 NR e
10e91 a
Thiacloprid N 1.8; 10 1 1.9e7.8 1 f
8 NR c

Class: H, herbicide; F, fungicide; N, neonicotinoid; O, organophosphate; P, pyrethroid. LOD, limit of detection; NR, not reported.
References: a, Calatayud-Vernich et al., 2016; b, Chauzat and Faucon, 2007; c, Johnson et al., 2010; d, Lodesani et al., 2008; e, Medici et al., 2012; f, Mullin et al., 2010; g, Serra-
Bonvehí and Orantes-Bermejo, 2010.

5. Conclusions agricultural pesticides and veterinary substances. The RB samples

had higher levels than HC samples for the xenobiotics chlorpyrifos-
These Uruguayan beeswax samples showed pollution with ethyl and tebuconazole. Additionally, the concentrations of
82 J. Harriet et al. / Chemosphere 177 (2017) 77e83
azoxystrobin, coumaphos, and tebuconazole in RB samples were
greater than in HC samples. These results suggest three imple-
mentation goals: (i) prioritization of HC as the main wax source for
the manufacture of new printed beeswax sheets; (ii) imple-
mentation of a risk assessment system to evaluate the biological
impacts of xenobiotics on beekeeping and (iii) the need for devel-
oping land planning strategies to reduce the environmental im-
pacts of agricultural production systems on beekeeping.
Acknowledgments
The chemical analysis of this work was funded by the companies
Apícola Integral Las Piedras (AILP) and the Honey Exporter Asso-
ciation from Uruguay (ADEXMI). The authors thank the Uruguayan
companies AILP and TELGAR for the beeswax samples. Finally, we
thank the National Honorary Commission for Beekeeping Devel-
opment (CHDA), which sponsored this study.
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